The present invention relates to processes of enhancing plant growth and productivity and more specifically, to the field of carbon re-allocation in plants.
Increasing harvestable plant yield is a major goal of all plant breeding efforts. In fiber producing crops, the economic value of this yield is directly related to the amount, location, and length of the cellulose fibers. It has been suggested that cellulose content and fiber yield is limited by the amount of substrate, or sugars, produced during photosynthesis. However, numerous studies provide evidence that although crucial for plant growth and survival, the availability of carbohydrates derived from photosynthesis are not major limiting factors in cellulose synthesis. Thus there exists a substantial opportunity to increase fiber yield by creating a sink for this existing photosynthate in cells high in cellulose. Sucrose, the major form of translocatable carbohydrate produced during photosynthesis in the plant, is translocated to sink tissue where it is converted to other compounds such as starch or cellulose.
Despite the fact that the amount of photosynthates in the plant are not a primary limitation in cellulose content, the rate of photosynthesis plays a large role in the overall growth of a plant. Further, one element in the control of photosynthesis in the plant is the feedback-inhibition of photosynthesis by photosynthetic products, such as starch, sucrose and hexose sugars. Goldschmidt and Huber (1992)1 tested the effect of girdling the leaves of crop plants and demonstrated that the build up of starch and other products of photosynthesis actually inhibited the rate of photosynthesis. These findings, and others (Sonnewald and Willmitzer 1992)2, indicate the photosynthetic rate, and ultimately plant growth, may be directly correlated with the rate that photosynthates are drawn away from the leaf, or the rate of biosynthetic degradation in the leaves. The degradation of photosynthates occurs primarily in cells/tissues that are actively growing (meristematic or young tissues) or in tissues where photosynthates are utilized for storage or structural components (sink tissues). Therefore altering the rate that carbohydrates are translocated to these sink tissues (altering carbon allocation) would not only increase overall plant growth (remove inhibitors of photosynthesis), but also increase the amount of storage (starch) or structural components (cellulose).
A striking example of the benefits of altering carbon allocation has been demonstrated in potato. By increasing the synthesis and accumulation of ADP-glucose in the tuber, starch synthesis increased which significantly increased dry matter content. In fact, this resulted in a 25% increase in tuber yield. The increase in ADP-glucose in the tuber was accomplished by genetically engineering the potato with a bacterial ADP-glucose pyrophosphorylase gene controlled by a tuber specific promoter (Shewmaker and Stalker 1992)3.
Much like ADP-glucose is a precursor to starch synthesis, the nucleotide sugar UDP-Glucose, (UDPG), is a high energy substrate for cellulose biosynthesis in both bacteria and higher plants (Delmer 19874. Delmer et al. 19955). Several bacterial genes which encode the enzyme UDP-glucose pyrophosphorylase (UDPG-PPase), responsible for the synthesis of UDPG, have been isolated (Ross et al. 19916). An existing patent by Betlach (19877) claims increased synthesis of xanthan and other polysaccharides in bacteria by insertion of a UDPG-PPase gene from Xanthamonas campestris. However, the claims in this patent are limited to increasing polysaccharide biosynthesis in prokaryotic organisms.
It is an object of the present invention to obviate or mitigate the above disadvantages.
The present invention provides a process of increasing plant growth and yield which comprises introducing into a plant a DNA sequence encoding a product which modifies, in the plant, the level of cellulose precursors. This product of the present invention includes, but is not limited to, ribonucleic acid (xe2x80x9cRNAxe2x80x9d) molecules, enzymes related to cellulose biosynthesis and proteins which regulate the expression of these enzymes.
It has been found that the process of the present invention leads to the reallocation by simple diffusion of carbohydrates such as glucose from photosynthetic cells, such as the leaf cells, to other cells within the plant. This translocation removes the inhibition on photosynthesis imposed by excess photosynthate accumulation in these photosynthetic cells thereby allowing the plant to produce more simple sugars by continued photosynthesis. In other words, as photosynthesis continues in an uninhibited fashion, more simple sugars are produced than would have otherwise have been possible. These simple sugars are building blocks for plant growth via the production of polymers such as starch and cellulose.
Further, the present invention provides a process of modifying the production of cellulose in a plant which comprises introducing into said plant a DNA sequence encoding a product which modifies, in the plant, the level of cellulose substrates. As above, the product includes, but is not limited to, ribonucleic acid (xe2x80x9cRNAxe2x80x9d) molecules, enzymes related to cellulose biosynthesis and proteins which regulate the expression of these enzymes.
The subject invention also provides a plant having increased growth and yield and/or modified cellulose producing activity as a result of introducing into said plant or parent of said plant a DNA sequence coding for a product which modifies the level of cellulose precursors in the plant.
Another aspect of the present invention provides for a DNA expression vector comprising a DNA sequence encoding a product which modifies, in a host, the level of cellulose precursors, said sequence being operably linked to an expression affecting DNA sequence and flanked by translational start and stop sequences.
The present invention also provides a genetically modified seed comprising a DNA sequence, said sequence encoding a product capable of increasing growth and yield and/or modifying the level of cellulose precursors in the plant resulting from said seed.
There are two primary features of the process of the present invention. Firstly, in all plants regardless of whether they are fiber-producing (trees, hemp, cotton etc..) or not, what is achieved are plants having faster rates of growth and increased yield by non-specifically re-allocating carbon within the plant away from photosynthetic cells. This allows photosynthesis to continue uninhibited to produce more simple xe2x80x9cconstructionxe2x80x9d sugars thereby enhancing the efficiency of the plant growth rate and increasing growth yield. Secondly, in fiber-producing plants, the expression of the DNA sequence introduced into the plant may be targeted to specific individual cell types within the plant to increase predictably cellulose deposition in a cell specific manner. In forest trees, this is expected to increase wood production and fiber yield, especially when the gene is linked to a promoter which expresses only in wood forming tissues. Increased fiber yield can also be expected in other non-forestry fiber producing plants, such as hemp and sisal. In addition to targeting wood forming tissues, increased cellulose production can be obtained in other parts of the plant such as the boles surrounding the seeds of cotton plants.
Specific applications for increased cellulose synthesis include numerous crops with diverse uses and growth habits. In forestry, wood production is influenced by a combination of physiological and biochemical processes governed by substantial genetic variation. This has lead to the theoretical consideration of limitations on increasing yield due to fundamental constraints on energy supply (Farnum 19838). Despite such limitations, increases in tree growth of 50 to 300% are possible depending on the tree species and growing environment. Clearly, improving energy capture, conversion of radiant energy, and altering carbon allocation within the plant are promising areas for tree improvement. Increasing cellulose content by the processes outlined herein can achieve such gains.